Introduction

Presently, hundreds of monoclonal antibodies (mAbs) and mAb fragments are under clinical development because of their excellent potential for the systemic treatment of cancer and other pathological conditions (1, 2). Positron emission tomography (PET) offers an exciting imaging option to confirm and quantify selective tumor uptake of such targeting molecules (3).

To enable PET imaging of mAbs (immuno-PET), an appropriate positron emitter, with a half-life (t1/2) that is compatible with the time needed to achieve optimal tumor-to-nontumor ratios (typically 2-4 days for intact mAbs), has to be securely coupled to the targeting molecule. For this purpose we recently described the large scale production of pure zirconium-89 (89Zr; t1/2: 78.4 h) and a strategy for labeling mAbs with 89Zr via a multi-step synthesis using a succinylated-derivative of desferrioxamine B (Df) as bifunctional chelate (4). The utility of this approach was clearly demonstrated through high quality 89Zr-mAb-PET images reported in preclinical and clinical studies (5-11). The upcoming commercialization of 89Zr will make this radionuclide generally available for research and clinical development.

A shortcoming of the aforementioned approach is that the multi-step procedure is relatively complicated and time consuming. In this updated protocol we present the newly developed p-isothiocyanatobenzyl-derivative of Df (SCN-Bz-Df; Macrocyclics, TX) which provides an efficient and rapid preparation of 89Zr-labeled mAbs. First, SCN-Bz-Df is coupled to the amine groups of a protein at pH 9.0, followed by purification using gel filtration. Next, the conjugate is labeled at room temperature by addition of a solution of 89Zr oxalic acid followed by purification using gel filtration. SCN-Bz-Df can also be utilized to label proteins at room temperature with gallium-68 (68Ga; t1/2: 1.13 h). 68Ga is especially attractive for PET-imaging of fast kinetic targeting proteins like mAb fragments.

Procedure

This procedure may be scaled up or down, maintaining the same molar ratios of reagents. A schematic representation of the procedure is shown in Figure 1.

Conjugation reaction

Pipette the required amount of protein solution (max. 1 ml; by preference between 2 and 10 mg ml protein) into an eppendorf tube. Adjust the reaction mixture to a total volume of 1 ml by adding a sufficient amount of normal saline into the tube.

CRITICAL STEP Alternatively, the desired pH for the reaction can be obtained by adding a stronger sodium carbonate buffer or by dialyzing the protein stock solution against 0.1 M sodium bicarbonate buffer (pH 9.0).

Dissolve SCN-Bz-Df in DMSO at a concentration of between 2 and 5 mM (1.5-3.8 mg ml-1) depending on the amount of protein or antibody used. Add this to the protein solution to give a 3-fold molar excess of the chelator over the molar amount of protein and mix immediately. Keep the DMSO concentration below 5% in the reaction mixture.

xiii. Analyze the purified radiolabeled protein by ITLC and HPLC. When the radiochemical purity is greater than 95% it is ready for storage at 4 °C or dilution in 0.9% NaCl/gentisic acid 5 mg ml-1 (pH = 4.9-5.3) for in vitro or in vivo studies. The radiolabeled protein should be stable in storage for at least several days.

CRITICAL STEP Gentisic acid is introduced during labeling and storage to prevent deterioration of the protein integrity by radiation. Consideration should also be given to assessment of the biological function of the protein after the conjugation and labeling reaction.

B. Purification of Df-protein and subsequent labeling with 68Ga.

i. Rinse a PD10 column with 20 ml 0.25 M ammonium acetate (pH = 5.5).

ii. Pipette the conjugation reaction mixture onto the column and discard the flow-through.

vii. While gently shaking, slowly add 0.2-1.0 ml of the modified protein (typically 0.5-2 mg) into the reaction vial. Adjust the reaction mixture to a total volume of 1.5 ml by adding a sufficient amount of 0.25 M ammonium acetate (pH = 5.5) into the tube.

CRITICAL STEP The pH of the labeling reaction should be in the range of 5-6.

Wash the cartridge with 2 ml 4 M HCl and subsequently dry by sucking air through the cartridge.

Elute with small fractions of Milli-Q water (50-100 µl). Collect the purified and concentrated 68Ga and measure the activity.

Timing

Steps 1-4: 45 min

Steps 5A i-iv: 15 min

Steps 5A v-xiii: 1.5 h

Steps 5B i-iv: 15 min

Steps 5B v-xiii: 20 min

Troubleshooting

See Table 1

Anticipated Results

Typically, 0.9-1.5 Df moieties are coupled per antibody or protein molecule. Radiolabeling of the Df-conjugated mAb with 89Zr will result in overall labeling yields of >85%. Resulting 89Zr-mAb conjugates are optimal with respect to radiochemical purity (>95% according to ITLC and analytical HPLC), immunoreactivity, and in vivo stability. A representative HPLC chromatogram and SDS-PAGE gel of a 89Zr-labeled mAb (150 kDa) is shown in Figure 2a and 2c, respectively.

Radiolabeling of the Df-conjugated protein with 68Ga will result in overall labeling yields of >90%. The radiochemical purity of 68Ga-labeled proteins is >97% according to ITLC and analytic HPLC. A representative HPLC chromatogram and SDS-PAGE gel of a 68Ga-labeled nanobody13-15 (31 kDa) is shown in Figure 2b and 2d, respectively.

The positron emitters 89Zr and 68Ga can be applied to assess normal biodistribution, and confirm and quantitate selective tumor uptake of mAbs, mAb-fragments, non-traditional antibody-like scaffolds or other proteins of interest in animal and clinical studies using PET-imaging. A representative 89Zr-immuno-PET image is shown in Figure 3.

Acknowledgements

This project was supported in part by grants from the European Union FP6 (LSHC-CT-2003-5032, STROMA) and the Dutch Technology Foundation (STW; VBC.6120). The authors want to thank the technical staff of BV Cyclotron and the Radionuclide Center for supply and processing of 89Zr.

PET images (coronal slices) were obtained at 24, 48, and 72 h after i.v. injection of 89Zr-mAb U36 (3.7 MBq, 100 µg mAb) with a double-crystal-layer high resolution research tomograph PET scanner. Images planes have been chosen where the right tumor is optimal visible. Tumors are indicated by arrows.